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Introduction

The protocols in this section describe the steps involved in differentiating neural stem cells (NSC) to neurons, astrocytes, and oligodendrocyte lineages in vitro. NSCs are self-renewing multipotent stem cells that can be proliferated in vitro in supportive culture systems such as Stempro® NSC SFM and can further be differentiated into downstream lineages. The protocols described are primarily optimized with NSCs derived from human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Some optimization in terms of reagent concentration and duration of in vitro differentiation is expected for NSCs from other species such as rat or mouse, as well as with NSCs derived from patient-specific iPSCs.

Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut™ D-MEM/F-12) at a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete medium. Freeze unused portions in aliquots.

Mix the following components under aseptic conditions. For larger volumes, increase the component amounts proportionally: If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of complete medium.

Component

Final concentration

Amount

KnockOut™ D-MEM/F-12

1X

97 mL

GlutaMAX™-I Supplement

2 mM

1 mL

bFGF (prepared as 100 μg/mL stock)

20 ng/mL

20 μL

EGF (prepared as 100 μg/mL stock)

20 ng/mL

20 μL

StemPro® Neural Supplement

2%

2 mL

Note: You may observe a white precipitate when thawing StemPro® Neural Supplement; this precipitate will disappear when the supplement is completely thawed or dissolved.

Neural differentiation medium

Neural differentiation medium requires supplementation of Neurobasal® medium with B-27® Serum-Free Supplement and GlutaMAX™-I. Neural differentiation medium is stable for 2 weeks when stored in the dark at 2–8°C.

Oligodendrocyte differentiation medium

Oligodendrocyte differentiation medium requires supplementation of Neurobasal® medium with B-27®, GlutaMAX™-I, and T3. Oligodendrocyte differentiation medium is stable for 2 weeks when stored in the dark at 2–8°C.

Preparing matrix

Coating culture vessels with CELLstart™ substrate

Coat the surface of the culture vessel with the working solution of CELLStart™ CTS® (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).

Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 in air for 1 hour.

Remove the vessel from the incubator and store it until use. Immediately before use, remove all CELLStart™ CTS® solution and replace it with complete StemPro® NSC SFM.

Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with Parafilm®, for up to 2 weeks. Do not remove CELLStart™ CTS® solution until just prior to use. Make sure the plates do not dry out.

Coating culture vesselswith Geltrex® matrix

Thaw the Geltrex® matrix bottle at 4°C overnight to prevent polymerization. The next day, dilute Geltrex® matrix 1:2 with D-MEM/F-12 at 4°C to make 100X stock solution, using an ice bucket to keep the bottles cold. Quickly prepare 0.5-mL aliquots in 50-mL conical tubes (pre-chilled on ice), and store the tubes at –20°C.

Cover the whole surface of each culture plate with the Geltrex® matrix solution (1.5 mL for a 35-mm dish, 3 mL for 60-mm dish, 5 mL for a T-25 culture flask).

Seal each dish with Parafilm® to prevent drying, and incubate 1 hour at room temperature in a laminar flow hood.

Immediately before use, remove all Geltrex® matrix solution, wash once with D-PBS with calcium and magnesium, and replace pre-warmed complete medium.

Note: You may store the Geltrex® matrix–treated dish at 4°C, wrapped tightly with Parafilm®, for up to 1 month. Do not remove Geltrex® matrix solution until just prior to use.

Coating culture vessels with poly-L-ornithine and laminin

Dissolve poly-L-ornithine in cell culture-grade distilled water to make 10 mg/mL stock solution (500X). Aliquot the solution and store it at –20°C until use.

Thaw the laminin slowly at 2–8°C and prepare 10 μg/mL working solution in cell culture-grade distilled water. Aliquot the working solution into polypropylene tubes, and store the tubes at –20°C until use. Avoid repeated freeze/thaw cycles.
Note: Laminin may form a gel if thawed too rapidly.

Dilute the poly-L-ornithine stock solution 1:500 in cell culture-grade distilled water to make 20 μg/mL working solution.

Coat the surface of the culture vessel (with or without cover slips) with the poly‑L‑ornithine working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).

Incubate the culture vessel overnight at 4°C or for 1 hour at 37°C.

Rinse the culture vessel twice with sterile water.

Coat the surface of the culture vessel (with or without cover slips) with the laminin working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35‑mm dish).

Incubate the culture vessel overnight at 4°C or for 2 hours at 37°C.

Rinse the culture vessel with D-PBS without calcium or magnesium, and store the vessel covered with D-PBS until use. Immediately before use, remove all D-PBS and replace it with complete StemPro® NSC SFM.
Note: You may coat the plates in advance and store them at room temperature, wrapped tightly with Parafilm®, for up to 1 week. Do not remove D-PBS until just prior to use. Make sure the plates do not dry out.

Differentiating neural stem cells

Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. If the cells reach 90% confluency, it might be necessary to split the cells at a 1:2 ratio. However, do not split the cells once they reach day 9–10 of differentiation when they can get damaged during the passaging process.

Proceed to staining, described below. You may store slides for up to 3–4 weeks in D-PBS at 4°C before staining. Do not allow slides to dry.

Staining cells

Incubate cells for 30–60 minutes in blocking buffer (5% serum of the secondary antibody host species, 1% BSA, 0.1% Triton®-X in D-PBS with Ca2+ and Mg2+).
Note: If you are using a surface antigen such as GalC, omit Triton®-X from the blocking buffer.

Remove the blocking buffer and incubate the cells overnight at 4°C with primary antibody diluted in 5% serum. Ensure that the cell surfaces are covered uniformly with the antibody solution.

Wash the cells 3X for 5 minutes with D-PBS containing Ca2+ and Mg2+ (if using a slide, use a staining dish with a magnetic stirrer).

Incubate the cells with fluorescence-labeled secondary antibody (5% serum in D-PBS with Ca2+ and Mg2+) in the dark at 37°C for 30–45 minutes.

Wash the cells 3X with D-PBS containing Ca2+ and Mg2+, and in the last wash, counter stain the cells with DAPI solution (3 ng/mL) for 5–10 minutes, and rinse with D-PBS.

If desired, mount using 3 drops of ProLong® Gold antifade reagent per slide and seal with the cover slip. You may store the slides in the dark at 4°C.

Expected results

A

B

C

D

Figure 1. Fluorescence images (20X) of Gibco® hNSCs that have been cultured in StemPro® NSC SFM for three passages, and then allowed to differentiate into neurons, oligodendrocytes, or astrocytes. Upon directed differentiation, cells start to lose the undifferentiated NSC marker, nestin, but stain positive for the differentiated cell type markers Dcx, GalC, and GFAP. Cells were stained for the undifferentiated NSC markers nestin (red) and SOX2 (green) prior to directed differentiation (Panel A). Cell were then differentiated into neurons and glial cells, and respectively stained for the neuronal marker Dcx (green) (Panel B), for the oligodendrocyte marker GalC (red) (Panel C), or for the astrocyte marker, GFAP (green) (Panel D). The nuclei were counterstained with DAPI (blue) in panels B–D.